Induction of superoxide by 12‐O‐tetradecanoylphorbol‐13‐acetate and thapsigargin, a non‐phorbol‐ester—type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: A permissive role for the arachidonic acid cascade in signal transduction

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Abstract
Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter—stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemical-induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol-ester—sensitive SENCAR mice demonstrated a time- and dose-dependent release of superoxide anion (4–6 nmol/106 cells) when stimulated by 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50–70%) by preincubation with 40 μM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a protein-kinase inhibitor. Alternatively, TPA-stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG-stimulated SENCAR MPs released a significant amount of superoxide (2–3 nmol/106 cells) that was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 μM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 μM 1-oleoyl-2-acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two-dimensional gel-electrophoretic analysis indicated that TPA-induced phosphorylation of a 47-kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol-ester— and non-phorbol-ester—type tumor promoters activate MPs in SENCAR mice; such a response may be “permissive” for additive (or synergistic) interactions with PKC-driven signal pathways.

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